1. Field of the Invention
The present invention relates to Xylitol dehydrogenase-inactivated and arabinose reductase-inhibited mutant of Candida tropicalis, method of producing high-yield of xylitol using the same, and xylitol produced thereby. More specifically, the present invention relates to a xylitol production method, in which an inconvenience that concentration of dissolved oxygen in a xylitol-producing medium should be maintained at a very low level can be eliminated through the use of a xylitol dehydrogenase-inactivated mutant of Candida tropicalis, and the production of byproduct arabitol, which is produced when using a biomass as a substrate and adversely affects the yield of xylitol, can be significantly reduced through the use of Candida tropicalis mutant ara-89 (KCTC 11136bp), having an inhibited activity of arabinose reductase converting arabinose to arabitol, thus increasing the production of xylitol, as well as xylitol produced by the method.
2. Description of the Prior Art
Xylitol, a five-carbon sugar alcohol, has high sweetening power, and thus is widely used as a functional sugar substitute for sugar. Because xylitol is found naturally in a variety of fruits or vegetables in very small amounts, the extraction of xylitol from fruits or vegetables is not industrially advantageous. For this reason, xylitol is produced by a chemical method of reducing a hemicellulose hydrolyzate rich in xylose. However, in the chemical method, it is difficult to isolate and purify xylose or xylitol from other hydrolyzates occurring in the hemicellulose moiety, a lot of costs are incurred and the yield of xylitol is as low as about 50-60%. Also, it is a high-temperature and high-pressure employing alkali, which causes problems of risk and waste.
In recent attempts to overcome such shortcoming, biological methods for producing xylitol have been actively studied. Korean Patent Registration No. 1999819 discloses a method of producing xylitol, in which new strain Candida tropicalis is cultured in a medium, containing xylose, a nitrogen source and inorganic salts, to convert xylose to xylitol. Also, PCT International Patent Publication No. WO88/05467 discloses a method of producing xylitol from a high concentration of xylose by culturing Candida guilliermondii under conditions employing limited aeration. Such biological methods can employ, as a raw material, a relatively low purity of xylose compared to the chemical methods and are safe and environment-friendly, because the processes themselves are carried out at room temperature and atmospheric pressure. Thus, studies focused on the production of xylitol by a variety of bacteria, yeasts, fungi and recombinant yeasts have been conducted to produce xylitol in high productivity and high yield (Winkelhausen, E. et al., J. Ferment. Bioeng. 86:1-14, 1998; Granström, T. B. et al., Appl. Microbiol. Biotechnol. 74:277-281, 2007). However, bacteria and recombinant yeast strains were not suitable for the production of xylitol, because they showed a weak metabolic activity of producing xylose or low efficiency. However, among yeast strains, Candida sp strains are known as strains suitable for the biological production of xylitol, because they show high xylitol productivity and yield compared to other microorganisms.
According to studies conducted to date, it is known that, as shown in FIG. 1, Candida sp strains, such as C. guillermondi, C. parapsilosis and C. tropicalis, convert xylose, absorbed from cells, to xylitol by xylose reductase, and converts xylitol to xylulose by xylitol dehydrogenase. Xylulose is then converted to xylulose-5-phosphate by xylulokinase, and xylulose-5-phosphate is used for the growth and maintenance of cells through a pentose phosphate pathway (see Laplace, J. M. et al., Appl. Microbiol. Biotechnol., 36:158-162, 1991; Hahn-Hagerdal, B. et al., Enzyme Microb. Technol., 16:933-943, 1994). That is, xylitol-producing strains use xylose as a carbon source for cell growth. Xylitol converted from xylose by xylose reductase is then converted to xylulose by xylitol dehydrogenase, and in this case, when the aeration of the medium is limited such that the concentration of dissolved oxygen is maintained as low as 0.5-2.0%, intracellular redox imbalance is incurred to reduce the supply of nicotinamide adenine dinucleotide (NAD) as a cofactor required by xylitol dehydrogenase and inhibit the conversion of xylitol to xylulose. As a result, xylitol is accumulated in cells and media, and thus xylitol is produced from xylose in a yield of 60-80%. That is, in the prior technology of producing xylitol using xylitol-producing strains, it is necessary to limit aeration to maintain the concentration of dissolved oxygen at a low level. Thus, studies focused on enhancing the productivity and yield of xylitol by limiting the aeration of medium to maintain the concentration of dissolved oxygen at a low level so as to intentionally induce intracellular redox imbalance have been actively conducted (see Kim, S. Y. et. al., J. Ferment. Bioeng., 83(3):267-270, 1997; Korean Patent Publication No. 1996-030577).
Japanese Patent No. 1998276791 discloses a method of producing xylitol using a concentrated Candida parapsilosis strain at a dissolved oxygen concentration of 0.1-5.0%. However, when xylitol is produced using an industrial scale fermenter, it is substantially impossible to control the concentration of dissolved oxygen to a low level as described above, and even if the control is possible, the production yield is only about 70-80%. Also, there is processing inconvenience, as Korean Patent Registration No. 10-0259470 suggests an agitation rate for controlling oxygen transfer rate for optimizing xylitol production to microaerobic conditions corresponding to a DOT (percent concentration of oxygen in medium) of less than 1%.
Paying attention to the fact that xylitol produced in Candida sp strains is reduced by its conversion to xylulose by xylitol dehydrogenase, the present inventors have developed xylitol dehydrogenase-inactivated Candida tropicalis mutant, in which xylitol produced from xylose can no longer be used for cell growth, and thus xylose can be bioconverted to xylitol with a theoretical yield of 100% (Ko, B. S. et. al., Appl. Environ. Microbiol. 72:4207-4213, 2006). Thus, the present inventors filed an application for the protection of an invention relating to a method of producing xylitol in high yield using a xylitol-producing strain, in which the expression of xylitol dehydrogenase is inhibited (Korean Patent Application No. 10-2005-0079751, filed on Aug. 30, 2005).
Also, according to said invention, xylitol yield should reach a theoretical yield of 100%, but when a biomass hydrolyzate containing a large amount of xylose is used directly as a substrate, byproduct arabitol that adversely affects xylitol crystallization as the final step of the production process will be produced in large amounts, and xylitol productivity and final xylitol concentration will be so low that industrial economy cannot be achieved. For this reason, there is a need to develop to a novel technology that enhances productivity and minimizes the production of byproducts.